银染增强的纳米金标记探针对微量核酸的检测

本研究利用银染增强的纳米金技术建立了一种简单快速的核酸定量方法.该方法基于纳米金与烷巯基修饰的寡核苷酸分子共价键合作用,将纳米微粒报告基团标记在与靶核酸一端序列互补的寡核苷酸上,同时生物素化修饰另一端互补序列.靶核酸与两段寡核苷酸探针杂交后,借亲和素固定在酶标板孔内,通过纳米金催化的银染放大效应产生高灵敏的识别信号,适时记录其吸光度值从而实现核酸分子的定量.该检测方法检测单链核酸分子的灵敏度达0.1 fM,双链分子为10 fM.     

纳米金标记核酸探针检测小反刍兽疫 病毒核酸的研究

试验旨在探讨利用纳米金标记寡核苷酸探针快速检测小反刍兽疫病毒核酸的方法。针对小反刍兽疫病毒N基因的高度保守区设计两条特异寡核苷酸探针,一条探针5’端修饰生物素,另一条探针3’端修饰巯基。巯基化的探针通过Au-S键连接到纳米金颗粒上。靶核酸两端分别与两条探针结合,形成"生物素化探针-靶核酸-纳米金探针"复合物,该复合物通过生物素-亲和素系统,固定在固相载体上,最后银染放大信号。通过肉眼观察法、光镜观察法、分光光度法分析银染灰度,从而间接测定靶核酸的量。初步检测PPRV核酸最低浓度达10fmol/L,所需时间约为1.5h。该方法灵敏度高、操作简单,为临床检测小反刍兽疫病毒核酸提供实验数据和技术支持。     

应用纳米金探针检测HSV-2的目视化基因芯片

本实验建立了一种应用金标链霉亲和素探针的目视化高灵敏度检测单纯疱疹病毒2型(HerpesSimplexVirus-2,HSV-2)的基因芯片。该芯片以HSV-2DNA聚合酶的高保守区为靶序列,设计HSV-2特异性引物和探针,通过PCR反应使扩增产物标记上生物素;氨基修饰的探针固定在活化的玻片上,与生物素标记的扩增产物杂交;利用生物素与链酶亲和素高亲合力的特性,加入纳米金标记的链酶亲和素后形成生物素-链酶亲和素-纳米金生物反应放大系统;银染反应后,达到目视化检测HSV-2效果。该HSV-2检测基因芯片能目视化检测出100fmol/L的HSV-2扩增产物,具有灵敏度高,低成本的特点,通过临床标本验证表明该芯片具有较高的准确性。     

应用纳米金探针检测HSV-2的目视化基因芯片

本实验建立了一种应用金标链霉亲和素探针的目视化高灵敏度检测单纯疱疹病毒2型(Herpes Simplex Virus-2, HSV-2)的基因芯片.该芯片以HSV-2 DNA 聚合酶的高保守区为靶序列,设计HSV-2特异性引物和探针,通过PCR反应使扩增产物标记上生物素;氨基修饰的探针固定在活化的玻片上,与生物素标记的扩增产物杂交;利用生物素与链酶亲和素高亲合力的特性,加入纳米金标记的链酶亲和素后形成生物素-链酶亲和素-纳米金生物反应放大系统;银染反应后,达到目视化检测HSV-2效果.该HSV-2检测基因芯片能目视化检测出100fmol/L的HSV-2扩增产物,具有灵敏度高,低成本的特点,通过临床标本验证表明该芯片具有较高的准确性.     

基于纳米金的显色基因芯片快速检测病原微生物

为构建一种新型的比色芯片,以实现对临床常见感染病原微生物的快速、准确地检测和鉴定.采用纳米金标记巯基修饰的各待检病原微生物的特异性基因片段,与相应的以各待检靶序列的特异性寡核苷酸探针构建成的基因芯片杂交,并通过结合银染反应将杂交信号被放大形成裸眼可见的显色信息来判读检测结果.该芯片技术检测时间短,最低检测值达 100fmol/L.操作方法简单,不需要特殊设备,能部分满足临床检测的通量要求,具有很好的临床应用前景.     

基于纳米金复合探针的基因芯片膜转印检测法

建立了一种基于纳米金复合探针的基因芯片膜转印核酸检测新方法。首先,用纳米金颗粒同时标记检测探针P2和两种长短不同且生物素化的信号探针 (T10,T40),其中检测探针与靶DNA 5￠端互补,两种信号探针起信号放大作用。当靶DNA分子存在时,芯片表面捕捉探针P1 (与靶DNA分子3￠端互补) 通过碱基互补配对原则结合靶DNA分子,将其固定于芯片上,同时检测探针通过与靶DNA 5￠端互补配对将纳米金复合探针结合于芯片表面,结果在芯片表面形成“三明治”结构,后通过链霉亲和素-生物素反应,使芯片表面对应有靶DNA分子的部位结合上碱性磷酸酶,最后利用BCIP/NBT显色系统使芯片表面信号结果镜面转印至尼龙膜表面。当检测探针和信号探针摩尔比为1∶10,T10和T40摩尔比为9:1时可以检测1 pmol/L合成靶DNA分子或0.23 pmol/L结核分枝杆菌16S rDNA PCR扩增产物,检测结果通过普通的光学扫描仪读取或肉眼直接判读信号有无。本芯片检测系统灵敏度高,操作方法简单、快速,不需要特殊仪器设备,在生物分子的检测方面具有较高的应用价值。     

DNA功能化纳米金结合基因芯片可视化检测microRNAs

开发了一种纳米金探针结合基因芯片用于micro RNAs(mi RNAs)检测的高灵敏度便携式生物传感器.靶标mi RNAs特异性捕获探针固定在芯片上,通过与纳米金探针杂交进行检测,最后利用过氧化氢(H2O2)还原四氯金酸(HAu Cl4)来放大检测信号.利用单个纳米金探针结合基因芯片可以检测到10 pmol/L的靶标mi RNAs,测得mi R-126在胎牛血清中的回收率为81.5%~109.1%.用这种生物传感器检测肺癌组织样本总RNA中的mi R-126,结果与定量PCR结果具有一致性.利用双纳米金探针进一步提高了检测灵敏度,可以检测到1 fmol/L的mi R-125a-5p.整个分析时间不超过1 h,并且实验结果可以用肉眼观察.这个平台可以同时检测肺癌相关mi R-126和mi R-125a-5p,并且具有费用低、快速和便捷的优势,有望用于mi RNAs的超灵敏可视化检测.     

纳米金基因芯片结合通用PCR同时检测多个病原性真菌

基于致病性真菌rDNA基因分型技术,构建采用通用引物的一次PCK法,并结合纳米金新型基因芯片检测系统实现一次对多个临床常见致病性酵母菌的检测分析.用生物素标记的通用引物扩增各真菌DNA片段并与基因芯片杂交,然后将链酶亲和素标记的纳米金结合到杂交体上,杂交信号通过银染反应被放大形成裸眼可见的显色信息.结果表明,通用引物可扩增临床常见的真菌DNA,选用的各探针特异性强、可靠性好,芯片的最低检测值达50 fmol/L.临床样品检测结果与常规鉴定方法结果一致.该技术检测时间短、操作简单、不需要特殊设备,能部分满足临床检测的通量要求,具有很好的临床应用前景.     

纳米金标记-逐步银染法快速检测金黄色葡萄球菌

将纳米金探针应用于目的核酸的检测,具有与PCR相当的灵敏度和特异性.本研究建立了一种可以在微孔板上快速检测金黄色葡萄球菌的纳米金标记-逐步银染法.该方法利用已包被链霉亲和素的微孔板,将PCR扩增的金黄色葡萄球菌nuc基因与生物素探针、纳米金探针形成的三明治杂交结构锚定其上,然后在低温下逐步银染显色,通过酶标仪检测放大的银染信号.这种纳米金标记-逐步银染法可以在显著降低非特异性背景信号的同时放大银染信号,检测金黄色葡萄球菌nuc基因的灵敏度为1 pmol/L,比常温一步银染法的灵敏度提高约102倍. 51例临床标本的检测结果与PCR法一致,与培养生化鉴定法的检测结果之间无显著性差异(P >0.05). 综上所述,本研究成功构建了金黄色葡萄球菌的纳米金标记-逐步银染法,在病原微生物的快速检测领域表现出广阔的发展潜力.     

一种简易SNP检测方法的建立

目的：建立一种快速、简单的SNP（Single Nucleotide Polymorphisms）检测方法。方法：设计带生物素标记的扩增引物对检测用具有单碱基差异的野生型和突变型靶序列分别进行扩增,然后通过紫外交联的方式将相应检测靶序列的探针固定在硝酸纤维素膜上,借助Taq酶完成膜上单引物延伸,从而对探针捕获的靶序列进行延伸固定在膜上,最后使用生物素．亲和素酶联显色（ABs-ELISA）反应肉眼观察结果。结果：阳性和阴性对照探针显示正常。野生型探针和突变型探针能够分别特异性结合靶序列,并通过生物素和亲和索显色系统放大为一种肉眼可判断结果的检测形式。结论：建立了一种基于硝酸纤维素膜载体上进行核酸扩增的SNP检测方法。     

用生物传感器方法测定正常小鼠肝脏中miR-21活性

目的:建立生物传感器检测小鼠肝脏中microRNA (miRNA)活性的方法,测定miR-21在正常小鼠肝脏中的活性。方法:首先,分子克隆方法构建检测miRNA活性的通用型质粒传感器Dsensor。将各种miRNA的互补序列插入Dsensor中构建成相应miRNA活性检测传感器。用水动力法将检测miR-21的Dsensor注射至正常小鼠体内,以miR-122 Dsensor为阳性对照,miR-206 Dsensor为阴性对照,以不插入任何miRNA互补序列的Dsensor为空白对照。不同时间点尾静脉采血测定Gluc表达,处死小鼠测定肝脏中Fluc表达,比较计算得出miRNA活性。最后,用QRT-PCR法测定小鼠肝脏组织中miR-21、miR-122和miR-206表达水平。结果:用RIF(Relative inhibiting fold)值表示miRNA活性,miR-21、miR-122和miR-206活性分别为80.03±21.25,29.90±5.90和0.92±0.29,表明小鼠正常肝脏中miR-21的负调控活性明显高于miR-122。然而,QRT-PCR法测定结果显示,miR-21的表达水平明显低于miR-122,而miR-206几乎没有表达。从miR-21与miR-122的活性与表达水平比较发现,miR-21的表达水平并没有真实反映其活性。结论:成功建立了一种小鼠肝脏中miRNA活性检测方法;首次发现小鼠正常肝脏中miR-21活性水平比miR-122更高,而表达水平却明显低于miR-122。提示miR-21对于维持肝脏的正常生理功能的重要作用,值得进一步研究其调控的靶基因和机理。     

Two-tiered approach identifies a network of cancer and liver disease-related genes regulated by miR-122

MicroRNAs function as important regulators of gene expression and are commonly linked to development, differentiation, and diseases such as cancer. To better understand their roles in various biological processes, identification of genes targeted by microRNAs is necessary. Although prediction tools have significantly helped with this task, experimental approaches are ultimately required for extensive target search and validation. We employed two independent yet complementary high throughput approaches to map a large set of mRNAs regulated by miR-122, a liver-specific microRNA implicated in regulation of fatty acid and cholesterol metabolism, hepatitis C infection, and hepatocellular carcinoma. The combination of luciferase reporter-based screening and shotgun proteomics resulted in the identification of 260 proteins significantly down-regulated in response to miR-122 in at least one method, 113 of which contain predicted miR-122 target sites. These proteins are enriched for functions associated with the cell cycle, differentiation, proliferation, and apoptosis. Among these miR-122-sensitive proteins, we identified a large group with strong connections to liver metabolism, diseases, and hepatocellular carcinoma. Additional analyses, including examination of consensus binding motifs for both miR-122 and target sequences, provide further insight into miR-122 function.     

Chip-based visual detection of microRNA using DNA-functionalized gold nanoparticles

In the present study, we developed a highly sensitive and convenient biosensor consisting of gold nanoparticle(Au NP) probes and a gene chip to detect micro RNAs(mi RNAs). Specific oligonucleotides were attached to the glass surface as capture probes for the target mi RNAs, which were then detected via hybridization to the Au NP probes. The signal was amplified via the reduction of HAu Cl4 by H2O2. The use of a single Au NP probe detected 10 pmol L?1 of target mi RNA. The recovery rate for mi R-126 from fetal bovine serum was 81.5%–109.1%. The biosensor detection of mi R-126 in total RNA extracted from lung cancer tissues was consistent with the quantitative PCR(q PCR) results. The use of two Au NP probes further improved the detection sensitivity such that even 1 fmol L?1 of target mi R-125a-5p was detectable. This assay takes less than 1 h to complete and the results can be observed by the naked eye. The platform simultaneously detected lung cancer related mi R-126 and mi R-125a-5p. Therefore, this low cost, rapid, and convenient technology could be used for ultrasensitive and robust visual mi RNA detection.     

Retracted: Long noncoding LINC01551 promotes hepatocellular carcinoma cell proliferation,migration, and invasion by acting as a competing endogenous RNA of microRNA-122-5p to regulate ADAM10 expression

Hepatocellular carcinoma (HCC) is a severe disease with high mortality in the world. It has been shown that long noncoding RNA (lncRNA) might play a role in HCC. The aim of the present study was to identify the role of long intergenic noncoding RNA 01551 (LINC01551) in the HCC development and explore the underlying mechanism of LINC01551/miR-122-5p/ADAM10 axis. The differentially expressed lncRNAs associated with HCC were screened out by a microarray analysis. The expression of LINC01551, miR-122-5p, and ADAM10 was determined in HCC tissues and cells. The potential miRNA (miR-122-5p) regulated by LINC01551 was explored, and the target relationship between miR-122-5p and ADAM10 was confirmed. To evaluate the effect of LINC01551 and miR-122-5p on proliferation, migration, invasion, and apoptosis of HCC, different plasmids were delivered into MHCC97-H cells. High expression of LINC01551 and ADAM10 yet low-expression of miR-122-5p were revealed in HCC tissues and cells. Overexpression of miR-122-5p could downregulate ADAM10. Biological prediction websites and fluorescence in situ hybridization assay verified that LINC01551 was mainly expressed in the cytoplasm. Silencing LINC01551 reduced HCC cell viability, proliferation, migration, invasion, and cell cycle entry yet induce cell apoptosis. Upregulation of LINC01551 increased its ability of competitively binding to miR-122-5p, thus reducing miR-122-5p and upregulating ADAM10 expression, as well as promoting the proliferative, migrative, and invasive ability. Taken together the results, it is highly possible that LINC01551 functions as an competing endogenous RNA (ceRNA) to regulate the miRNA target ADAM10 by sponging miR-122-5p and therefore promotes the development of HCC, highlighting a promising competitive new target for the HCC treatment.     

MicroRNA-122a functions as a novel tumor suppressor downstream of adenomatous polyposis coli in gastrointestinal cancers

Aberrant regulation of APC/β-catenin signaling pathway is common in the pathogenesis of colorectal and other cancers. Targets regulated by APC/β-catenin signaling pathway play crucial roles in cancer development. In the current study, we aimed to illustrate the influence of APC/β-catenin signaling pathway on expression of microRNAs, one new group of players important to carcinogenesis. Restoration of APC function in colorectal cancer cells led to the deregulation of several cancer-related microRNAs, such as miR-122a which was recognized as the liver-specific microRNA. MiR-122a was down-regulated in gastrointestinal cancer cell lines as well as primary carcinoma tissues. Inhibition of miR-122a could reverse wild-type APC-induced growth inhibition of gastrointestinal cancer cells while miR-122a mimic inhibited cell growth. In summary, we identified some cancer-related microRNAs regulated by APC/β-catenin signaling pathway. The down-regulation of miR-122a mediated by aberrant APC/β-catenin signaling is important to the pathogenesis of gastrointestinal cancers.     

Upregulated lncRNA-UCA1 contributes to metastasis of bile duct carcinoma through regulation of miR-122/CLIC1 and activation of the ERK/MAPK signaling pathway

In the present study, we aimed to identify specific lncRNAs and miRNAs, as well as mRNAs, involved in bile duct carcinoma (BDC) and to further explore the way in which lncRNA UCA1 regulates cell metastasis ability. Differentially expressed RNAs were screened out from the TCGA database. In in vitro experiments, qRT-PCR was used to measure lncRNA UCA1, miR-122 and CLIC1 expression. We performed a dual luciferase assay to validate the target relationships among UCA1, CLIC1 and miR-122. The cell migration ability was measured by a wound healing assay, and Transwell assays were applied to detect cell invasive ability. Western blot analysis was employed to detect the expression of related proteins in the MAPK signaling pathway. According to the bioinformatics analysis, lncRNA UCA1 and CLIC1 were both significantly upregulated in BDC, while the expression of miR-122 declined compared with the normal group. The target relationship among UCA1, CLIC1 and miR-122 was verified. UCA1 promoted BDC cell migration and invasiveness, while miR-122 inhibited their progression. CLIC1 served as the downstream target gene of miR-122 and had opposite effects. The ERK/MAPK signaling pathway was activated after upregulating UCA1. LncRNA-UCA1 promoted the metastasis of BDC cells by regulating the expression of miR-122 and its downstream gene mRNA CLIC1 and promoted the activation of the ERK/MAPK pathway, which expanded the horizons of targeted therapy of cholangiocarcinoma.     

Regulation and biological function of the liver-specific miR-122

miRNAs (microRNAs) are important regulators of gene expression. In higher eukaryotes, the tightly controlled expression of different miRNAs, each of which regulates multiple target mRNAs, is crucial for the maintenance of tissue type and the control of differentiation. miR-122 is a highly liver-specific miRNA that is important in hepatitis C virus infection, cholesterol metabolism and hepatocellular carcinoma. In the present review, we discuss the effects of miR-122 on liver physiology and pathology. Recent evidence of pathways involved in the regulation of miR-122 expression is also considered.